Moving Beyond Isothiocyanates: A Look at the Stability of Conjugation Links Toward Radiolysis in 89Zr-Labeled Immunoconjugates.

Zirconium-89 is the most widely used radioisotope for immunoPET because its physical half-life (78.2 h) suits the one of antibodies. Desferrioxamine B (DFO) is the standard chelator for the complexation of zirconium(IV), and its bifunctional version, containing a phenylisothiocyanate function, is the most commonly used for the conjugation of DFO to proteins. However, preliminary results have shown that the thiourea link obtained from the conjugation of isothiocyanate and lysines is sensitive to the ionizing radiation generated by the radioisotope, leading to the rupture of the link and the release of the chelator/radiometal complex. This radiolysis phenomenon could produce nonspecific signal and prevent the detection of bone metastasis, as free zirconium accumulates into the bones. The aim of this work was to study the stability of a selection of conjugation linkers in 89Zr-labeled immunoconjugates. We have synthesized several DFO-based bifunctional chelators appended with an isothiocyanate moiety, a bicyclononyne, or a squaramate ester. Two antibodies (trastuzumab and rituximab) were conjugated and radiolabeled with zirconium-89. The effect of increasing activities of zirconium-89 on the integrity of the bioconjugate bearing thiourea links was evaluated as well as the impact of the presence of a radioprotectant. The stability of the radiolabeled antibodies was studied over 7 days in PBS and human plasma. Radioconjugates' integrity was evaluated using iTLC and size-exclusion chromatography. This study shows that the nature of the linker between the chelator and biomolecule can have a strong impact on the stability of the 89Zr-labeled conjugates, as well as on the aggregation of the conjugates.

Diagnostic Positron Emission Tomography Imaging with Zirconium-89 Desferrioxamine B Squaramide: From Bench to Bedside.

ConspectusMolecular imaging with antibodies radiolabeled with positron-emitting radionuclides combines the affinity and selectivity of antibodies with the sensitivity of Positron Emission Tomography (PET). PET imaging allows the visualization and quantification of the biodistribution of the injected radiolabeled antibody, which can be used to characterize specific biological interactions in individual patients. This characterization can provide information about the engagement of the antibody with a molecular target such as receptors present in elevated levels in tumors as well as providing insight into the distribution and clearance of the antibody. Potential applications of clinical PET with radiolabeled antibodies include identifying patients for targeted therapies, characterization of heterogeneous disease, and monitoring treatment response.Antibodies often take several days to clear from the blood pool and localize in tumors, so PET imaging with radiolabeled antibodies requires the use of a radionuclide with a similar radioactive half-life. Zirconium-89 is a positron-emitting radionuclide that has a radioactive half-life of 78 h and relatively low positron emission energy that is well suited to radiolabeling antibodies. It is essential that the zirconium-89 radionuclide be attached to the antibody through chemistry that provides an agent that is stable in vivo with respect to the dissociation of the radionuclide without compromising the biological activity of the antibody.This Account focuses on our research using a simple derivative of the bacterial siderophore desferrioxamine (DFO) with a squaramide ester functional group, DFO-squaramide (DFOSq), to link the chelator to antibodies. In our work, we produce conjugates with an average ∼4 chelators per antibody, and this does not compromise the binding of the antibody to the target. The resulting antibody conjugates of DFOSq are stable and can be easily radiolabeled with zirconium-89 in high radiochemical yields and purity. Automated methods for the radiolabeling of DFOSq-antibody conjugates have been developed to support multicenter clinical trials. Evaluation of several DFOSq conjugates with antibodies and low molecular weight targeting agents in tumor mouse models gave PET images with high tumor uptake and low background. The promising preclinical results supported the translation of this chemistry to human clinical trials using two different radiolabeled antibodies. The potential clinical impact of these ongoing clinical trials is discussed.The use of DFOSq to radiolabel relatively low molecular weight targeting molecules, peptides, and peptide mimetics is also presented. Low molecular weight molecules typically clear the blood pool and accumulate in target tissue more rapidly than antibodies, so they are usually radiolabeled with positron-emitting radionuclides with shorter radioactive half-lives such as fluorine-18 (t1/2 ∼ 110 min) or gallium-68 (t1/2 ∼ 68 min). Radiolabeling peptides and peptide mimetics with zirconium-89, with its longer radioactive half-life (t1/2 = 78 h), could facilitate the centralized manufacture and distribution of radiolabeled tracers. In addition, the ability to image patients at later time points with zirconium-89 based agents (e.g. 4-24 h after injection) may also allow the delineation of small or low-uptake disease sites as the delayed imaging results in increased clearance of the tracer from nontarget tissue and lower background signal.

Electrospun Biomimetic Periosteum Capable of Controlled Release of Multiple Agents for Programmed Promoting Bone Regeneration.

The effective repair of large bone defects remains a major challenge due to its limited self-healing capacity. Inspired by the structure and function of the natural periosteum, an electrospun biomimetic periosteum is constructed to programmatically promote bone regeneration using natural bone healing mechanisms. The biomimetic periosteum is composed of a bilayer with an asymmetric structure in which an aligned electrospun poly(ε-caprolactone)/gelatin/deferoxamine (PCL/GEL/DFO) layer mimics the outer fibrous layer of the periosteum, while a random coaxial electrospun PCL/GEL/aspirin (ASP) shell and PCL/silicon nanoparticles (SiNPs) core layer mimics the inner cambial layer. The bilayer controls the release of ASP, DFO, and SiNPs to precisely regulate the inflammatory, angiogenic, and osteogenic phases of bone repair. The random coaxial inner layer can effectively antioxidize, promoting cell recruitment, proliferation, differentiation, and mineralization, while the aligned outer layer can promote angiogenesis and prevent fibroblast infiltration. In particular, different stages of bone repair are modulated in a rat skull defect model to achieve faster and better bone regeneration. The proposed biomimetic periosteum is expected to be a promising candidate for bone defect healing.

Development of a [89Zr]Zr-labeled Human Antibody using a Novel Phage-displayed Human scFv Library.

PURPOSE: Tax-interacting protein 1 (TIP1) is a cancer-specific radiation-inducible cell surface antigen that plays a role in cancer progression and resistance to therapy. This study aimed to develop a novel anti-TIP1 human antibody for noninvasive PET imaging in patients with cancer. EXPERIMENTAL DESIGN: A phage-displayed single-chain variable fragment (scFv) library was created from healthy donors' blood. High-affinity anti-TIP1 scFvs were selected from the library and engineered to human IgG1. Purified Abs were characterized by size exclusion chromatography high-performance liquid chromatography (SEC-HPLC), native mass spectrometry (native MS), ELISA, BIAcore, and flow cytometry. The labeling of positron emitter [89Zr]Zr to the lead Ab, L111, was optimized using deferoxamine (DFO) chelator. The stability of [89Zr]Zr-DFO-L111 was assessed in human serum. Small animal PET studies were performed in lung cancer tumor models (A549 and H460). RESULTS: We obtained 95% pure L111 by SEC-HPLC. Native MS confirmed the intact mass and glycosylation pattern of L111. Conjugation of three molar equivalents of DFO led to the optimal DFO-to-L111 ratio of 1.05. Radiochemical purity of 99.9% and specific activity of 0.37 MBq/µg was obtained for [89Zr]Zr-DFO-L111. [89Zr]Zr-DFO-L111 was stable in human serum over 7 days. The immunoreactive fraction in cell surface binding studies was 96%. In PET, preinjection with 4 mg/kg cold L111 before [89Zr]Zr-DFO-L111 (7.4 MBq; 20 µg) significantly (P < 0.01) enhanced the tumor-to-muscle standard uptake values (SUVmax) ratios on day 5 compared with day 2 postinjection. CONCLUSIONS: L111 Ab targets lung cancer cells in vitro and in vivo. [89Zr]Zr-DFO-L111 is a human antibody that will be evaluated in the first in-human study of safety and PET imaging.

Chelation of Mitochondrial Iron as an Antiparasitic Strategy.

Iron, as an essential micronutrient, plays a crucial role in host-pathogen interactions. In order to limit the growth of the pathogen, a common strategy of innate immunity includes withdrawing available iron to interfere with the cellular processes of the microorganism. Against that, unicellular parasites have developed powerful strategies to scavenge iron, despite the effort of the host. Iron-sequestering compounds, such as the approved and potent chelator deferoxamine (DFO), are considered a viable option for therapeutic intervention. Since iron is heavily utilized in the mitochondrion, targeting iron chelators in this organelle could constitute an effective therapeutic strategy. This work presents mitochondrially targeted DFO, mitoDFO, as a candidate against a range of unicellular parasites with promising in vitro efficiency. Intracellular Leishmania infection can be cleared by this compound, and experimentation with Trypanosoma brucei 427 elucidates its possible mode of action. The compound not only affects iron homeostasis but also alters the physiochemical properties of the inner mitochondrial membrane, resulting in a loss of function. Furthermore, investigating the virulence factors of pathogenic yeasts confirms that mitoDFO is a viable candidate for therapeutic intervention against a wide spectrum of microbe-associated diseases.

Site-Specific 68Ga Radiolabeling of Trastuzumab Fab via Methionine for ImmunoPET Imaging.

Bioconjugates of antibodies and their derivatives radiolabeled with ß+-emitting radionuclides can be utilized for diagnostic PET imaging. Site-specific attachment of radioactive cargo to antibody delivery vectors provides homogeneous, well-defined immunoconjugates. Recent studies have demonstrated the utility of oxaziridine chemistry for site-specific labeling of methionine residues. Herein, we applied this approach to site-specifically radiolabel trastuzumab-derived Fab immunoconjugates with 68Ga, which can be used for in vivo PET imaging of HER2-positive breast cancer tumors. Initially, a reactive azide was introduced to a single solvent-accessible methionine residue in both the wild-type Fab and an engineered derivative containing methionine residue M74, utilizing the principles of oxaziridine chemistry. Subsequently, these conjugates were functionalized with a modified DFO chelator incorporating dibenzocyclooctyne. The resulting DFO-WT and DFO-M74 conjugates were radiolabeled with generator-produced [68Ga]Ga3+, to yield the novel PET radiotracers, [68Ga]Ga-DFO-WT and [68Ga]Ga-DFO-M74. In vitro and in vivo studies demonstrated that [68Ga]Ga-DFO-M74 exhibited a higher affinity for HER2 receptors. Biodistribution studies in mice bearing orthotopic HER2-positive breast tumors revealed a higher uptake of [68Ga]Ga-DFO-M74 in the tumor tissue, accompanied by rapid renal clearance, enabling clear delineation of tumors using PET imaging. Conversely, [68Ga]Ga-DFO-WT exhibited lower uptake and inferior image contrast compared to [68Ga]Ga-DFO-M74. Overall, the results demonstrate that the highly facile methionine-oxaziridine modification approach can be simply applied to the synthesis of stable and site-specifically modified radiolabeled antibody-chelator conjugates with favorable pharmacokinetics for PET imaging.

DFO-Km: A Modular Chelator as a New Chemical Tool for the Construction of Zirconium-89-Based Radiopharmaceuticals.

Zirconium-89-labeled monoclonal antibodies and other large macromolecules such as nanoparticles hold great promise as positron emission tomography imaging agents. In general, zirconium-89 is an ideal radionuclide for long-circulating vectors such as antibodies or nanoparticles. It is also a promising radionuclide for theranostic radiopharmaceuticals due to its suitable match in half-life with actinium-225, thorium-227, lutetium-177, and others. As such, demand for new and optimized bifunctional chelators for zirconium-89 continues to grow. Herein, we present the modular chelator DFO-Km, which is octadentate and features lysine as a modular amino acid linker. The modular amino acid linker can be changed to other natural or unnatural amino acids to access different bioconjugation chemistries, while the chelating portion is unchanged thus retaining identical metal ion coordination properties to DFO-Km. The epsilon-amine in the DFO-Km linker (lysine) was used to complete synthesis of a bifunctional derivative bearing a p-SCN-Ph moiety. The chelator DFO-Km includes a redesigned hydroxamic acid, which provides more flexibility for metal ion coordination relative to the monomer used in the previously published DFO-Em. Moreover, a set of comprehensive DFT calculations were performed to model and evaluate 16 geometric isomers of Zr-(DFO-Km), which suggested the complex would form the optimum cic-cis-trans-trans octadentate Zr(IV) coordination geometry with no aqua or hydroxide ligands present. The bifunctional derivative p-SCN-Ph-DFO-Km was compared directly with the commercially available p-SCN-Ph-DFO, and both underwent efficient conjugation to a nonspecific human serum antibody (IgG) to yield two model immunoconjugates. The behavior of [89Zr]Zr-DFO-Km-IgG was studied in healthy mice for 2 weeks and compared to an equivalent cohort injected with [89Zr]Zr-DFO-IgG as a clinical "gold standard" control. PET-CT and biodistribution results revealed higher stability of [89Zr]Zr-(DFO-Km)-IgG in vivo over [89Zr]Zr-DFO-IgG, as demonstrated by the significant reduction of zirconium-89 in the whole skeleton as visualized and quantified by PET-CT at 1, 3, 7, and 14 days post-injection. Using CT-gated regions of interest over these PET-CT images, the whole skeleton was selected and uptake values were measured at 14 days post-injection of 3.6 ± 0.9 (DFO) vs 1.9 ± 0.1 (DFO-Km) %ID/g (n = 4, * p = 0.02), which represents a ∼48% reduction in bone uptake with DFO-Km relative to DFO. Biodistribution experiments performed on these same mice following the 14 day imaging time point revealed bone (both tibia) uptake values of 3.7 ± 1.3 (DFO) vs 2.0 ± 0.6 (DFO-Km) %ID/g (n = 6, * p < 0.05), with the tibia uptake values in close agreement with whole-skeleton ROI PET-CT data. These results indicate that DFO-Km is an improved chelator for [89Zr]Zr4+ applications relative to DFO. The bifunctional chelator p-SCN-Ph-DFO-Km shows potential as a new chemical tool for creating bioconjugates using targeting vectors such as antibodies, peptides, and nanoparticles.

Visualizing Galectin-3 Binding Protein Expression with ImmunoPET.

Galectin-3 binding protein (Gal-3BP) is a glycoprotein that is overexpressed and secreted by several cancers and has been implicated as a marker of both tumor progression and poor prognosis in melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, and breast cancer. The expression of Gal-3BP by a variety of neoplasms makes it an enticing target for both diagnostics and therapeutics, including immuno-positron emission tomography (immunoPET) probes and antibody-drug conjugates (ADCs). Herein, we report the development, in vitro characterization, and in vivo evaluation of a pair of Gal-3BP-targeting radioimmunoconjugates for 89Zr-immunoPET. A humanized anti-Gal-3BP antibody, 1959, and its corresponding ADC, 1959-sss/DM4 (DM4 = ravtansine), were modified with desferrioxamine (DFO) to yield DFO-1959 and DFO-1959-sss/DM4 immunoconjugates bearing 1-2 DFO/monoclonal antibody. Both DFO-modified immunoconjugates retained their affinity for Gal-3BP in enzyme-linked immunosorbent assay experiments. The chelator-bearing antibodies were radiolabeled with zirconium-89 (t1/2 ≈ 3.3 d) to produce radioimmunoconjugates ─ [89Zr]Zr-DFO-1959 and [89Zr]Zr-DFO-1959-sss/DM4 ─ with high specific activity (>444 MBq/mg, >12 mCi/mg) and stability (>80% intact after 168 h in human serum at 37 °C). In mice bearing subcutaneous Gal-3BP-secreting A375-MA1 xenografts, [89Zr]Zr-DFO-1959 clearly delineated tumor tissue, reaching a maximum tumoral activity concentration (54.8 ± 15.8%ID/g) and tumor-to-background contrast (tumor-to-blood = 8.0 ± 4.6) at 120 h post-injection. The administration of [89Zr]Zr-DFO-1959 to mice bearing subcutaneous Gal-3BP-expressing melanoma patient-derived xenografts produced similarly promising results. [89Zr]Zr-DFO-1959 and [89Zr]Zr-DFO-1959-sss/DM4 exhibited nearly identical pharmacokinetic profiles in the mice bearing A375-MA1 tumors, though the latter produced higher uptake in the spleen and kidneys. Both [89Zr]Zr-DFO-1959 and [89Zr]Zr-DFO-1959-sss/DM4 effectively visualized Gal-3BP-secreting tumors in murine models of melanoma. These results suggest that both probes could play a role in the clinical imaging of Gal-3BP-expressing malignancies, particularly as companion theranostics for the identification of patients likely to respond to Gal-3BP-targeted therapeutics such as 1959-sss/DM4.

Evaluation of coumarin-tagged deferoxamine as a Zr(IV)-based PET/fluorescence dual imaging probe.

Desferoxamine (DFO) is currently the golden standard chelator for 89Zr4+, a promising nuclide for positron emission tomography imaging (PET). The natural siderophore DFO had previously been conjugated with fluorophores to obtain Fe(III) sensing molecules. In this study, a fluorescent coumarin derivative of DFO (DFOC) has been prepared and characterized (potentiometry, UV-Vis spectroscopy) for what concerns its protonation and metal coordination properties towards PET-relevant ions (Cu(II), Zr(IV)), evidencing strong similarity with pristine DFO. Retention of DFOC fluorescence emission upon metal binding has been checked (fluorescence spectrophotometry), as it would - and does - allow for optical (fluorescent) imaging, thus unlocking bimodal (PET/fluorescence) imaging for 89Zr(IV) tracers. Crystal violet and MTT assays on NIH-3 T3 fibroblasts and MDA-MB 231 mammary adenocarcinoma cell lines demonstrated, respectively, no cytotoxicity nor metabolic impairment at usual radiodiagnostic concentrations of ZrDFOC. Clonogenic colony-forming assay performed on X-irradiated MDA-MB 231 cells showed no interference of ZrDFOC with radiosensitivity. Morphological biodistribution (confocal fluorescence, transmission electron microscopy) assays on the same cells suggested internalization of the complex through endocytosis. Overall, these results support fluorophore-tagged DFO as a suitable option to achieve dual imaging (PET/fluorescence) probes based on 89Zr.

Synthesis of Modular Desferrioxamine Analogues and Evaluation of Zwitterionic Derivatives for Zirconium Complexation.

The natural siderophore desferrioxamine B (DFOB) has been used for targeted PET imaging with 89 Zr before. However, Zr-DFOB has a limited stability and a number of derivatives have been developed with improved chelation properties for zirconium. We describe the synthesis of pseudopeptidic analogues of DFOB with azido side chains. These are termed AZA-DFO (hexadentate) and AZA-DFO* (octadentate) and are assembled via a modular synthesis from Orn-ß-Ala and Lys-ß-Ala. Nine different chelators have been conjugated to zwitterionic moieties by copper-catalyzed azide-alkyne cycloaddition (CuAAC). The resulting water-soluble chelators form Zr complexes under mild conditions (room temperature for 90 min). Transchelation assays with 1000-fold excess of EDTA and 300-fold excess of DFOB revealed that a short spacing of hydroxamates in (Orn-ß-Ala)3-4 leads to improved complex stability compared to a longer spacing in (Lys-ß-Ala)3-4 . We found that the alignment of amide groups in the pseudopeptide backbone and the presence of zwitterionic sidechains did not compromise the stability of the Zr-complexes with our chelators. We believe that the octadentate derivative AZA-DFO* is particularly valuable for the preparation of new Zr-chelators for targeted imaging which combine tunable pharmacokinetic properties with high complex stability and fast Zr-complexation kinetics.

Site-Specific Photoaffinity Bioconjugation for the Creation of 89Zr-Labeled Radioimmunoconjugates.

PURPOSE: Site-specific approaches to bioconjugation produce well-defined and homogeneous immunoconjugates with potential for superior in vivo behavior compared to analogs synthesized using traditional, stochastic methods. The possibility of incorporating photoaffinity chemistry into a site-specific bioconjugation strategy is particularly enticing, as it could simplify and accelerate the preparation of homogeneous immunoconjugates for the clinic. In this investigation, we report the synthesis, in vitro characterization, and in vivo evaluation of a site-specifically modified, 89Zr-labeled radioimmunoconjugate created via the reaction between an mAb and an Fc-binding protein bearing a photoactivatable 4-benzoylphenylalanine residue. PROCEDURES: A variant of the Fc-binding Z domain of protein A containing a photoactivatable, 4-benzoylphenylalanine residue - Z(35BPA) - was modified with desferrioxamine (DFO), combined with the A33 antigen-targeting mAb huA33, and irradiated with UV light. The resulting immunoconjugate - DFOZ(35BPA)-huA33 - was purified and characterized via SDS-PAGE, MALDI-ToF mass spectrometry, surface plasmon resonance, and flow cytometry. The radiolabeling of DFOZ(35BPA)-huA33 was optimized to produce [89Zr]Zr-DFOZ(35BPA)-huA33, and the immunoreactivity of the radioimmunoconjugate was determined with SW1222 human colorectal cancer cells. Finally, the in vivo performance of [89Zr]Zr-DFOZ(35BPA)-huA33 in mice bearing subcutaneous SW1222 xenografts was interrogated via PET imaging and biodistribution experiments and compared to that of a stochastically labeled control radioimmunoconjugate, [89Zr]Zr-DFO-huA33. RESULTS: HuA33 was site-specifically modified with Z(35BPA)-DFO, producing an immunoconjugate with on average 1 DFO/mAb, high in vitro stability, and high affinity for its target. [89Zr]Zr-DFOZ(35BPA)-huA33 was synthesized in 95% radiochemical yield and exhibited a specific activity of 2 mCi/mg and an immunoreactive fraction of ~ 0.85. PET imaging and biodistribution experiments revealed that high concentrations of the radioimmunoconjugate accumulated in tumor tissue (i.e., ~ 40%ID/g at 120 h p.i.) but also that the Z(35BPA)-bearing immunoPET probe produced higher uptake in the liver, spleen, and kidneys than its stochastically modified cousin, [89Zr]Zr-DFO-huA33. CONCLUSIONS: Photoaffinity chemistry and an Fc-binding variant of the Z domain were successfully leveraged to create a novel site-specific strategy for the synthesis of radioimmunoconjugates. The probe synthesized using this method - DFOZ(35BPA)-huA33 - was well-defined and homogeneous, and the resulting radioimmunoconjugate ([89Zr]Zr-DFOZ(35BPA)-huA33) boasted high specific activity, stability, and immunoreactivity. While the site-specifically modified radioimmunoconjugate produced high activity concentrations in tumor tissue, it also yielded higher uptake in healthy organs than a stochastically modified analog, suggesting that optimization of this system is necessary prior to clinical translation.

New Long-Acting [89Zr]Zr-DFO GLP-1 PET Tracers with Increased Molar Activity and Reduced Kidney Accumulation.

Positron emission tomography (PET) imaging is used in drug development to noninvasively measure biodistribution and receptor occupancy. Ideally, PET tracers retain target binding and biodistribution properties of the investigated drug. Previously, we developed a zirconium-89 PET tracer based on a long-circulating glucagon-like peptide 1 receptor agonist (GLP-1RA) using desferrioxamine (DFO) as a chelator. Here, we aimed to develop an improved zirconium-89-labeled GLP-1RA with increased molar activity to increase the uptake in low receptor density tissues, such as brain. Furthermore, we aimed at reducing tracer accumulation in the kidneys. Introducing up to four additional Zr-DFOs resulted in higher molar activity and stability, while retaining potency. Branched placement of DFOs was especially beneficial. Tracers with either two or four DFOs had similar biodistribution as the tracer with one DFO in vivo, albeit increased kidney and liver uptake. Reduced kidney accumulation was achieved by introducing an enzymatically cleavable Met-Val-Lys (MVK) linker motif between the chelator and the peptide.

Quantitative PET tracking of intra-articularly administered 89Zr-peptide-decorated nanoemulsions.

Intra-articular (IA) administration of drugs for the treatment of diseases such as rheumatoid arthritis, osteoarthritis and psoriatic arthritis is a common strategy; however, the rapid clearance from the synovial fluid restricts their effectivity due to the limited retention time. Drug Delivery Systems (DDS) are currently being developed to increase their joint retention time. This study compares the biodistribution and retention time of a senolytic peptide (PEP), with potential application in osteoarthritis disease, and this senolytic peptide encapsulated in a DDS based on a lipid nanoemulsion (PEPNE) by using positron emission tomography (PET) imaging. To this aim, the PEP was conjugated with a chelating agent (DFO) and radiolabeled with zirconium-89 (89Zr). Then, [89Zr]-PEP was encapsulated in a novel nanoemulsion formulation, composed by vitamin E, sphingomyelin, and a lipid-PEG. Afterward, healthy rats were administered with either the [89Zr]-PEP or the [89Zr]-PEP-NE via IA injection and underwent PET scans at 0.5-, 24-, 48-, 72-, 168-, 240- and 336 h post-injection. To assess the biodistribution of both radiotracers, several volume-of-interest were manually drawn in different organs of the rat body and the %ID/organ was calculated. The [89Zr]-PEP was successfully encapsulated in the NE and their physicochemical properties were minimally affected by the radiolabeling buffer. Adequate stability of both [89Zr]-PEP and [89Zr]-PEP-NE was found in synovial fluid over 72 h. Quantitative data from PET images revealed a significantly higher [89Zr]-PEP-NE retention in the injected knee than with [89Zr]-PEP in all follow-up PET scans. The [89Zr]-PEP %ID/organ values in the liver and kidney were significantly higher than those from [89Zr]-PEP-NE, which might indicate a faster elimination of the [89Zr]-PEP. Therefore, the study highlights the higher retention time on the target site of the [89Zr]-PEP-NE which may improve the therapeutic effects of the peptide. Thereby, the novel nanoemulsion formulation seems to be a successful DDS for IA injection. In addition, these results represent the first study that evaluates the distribution of a PET-guided DDS after its IA administration.

Oral Non-absorbable Polymer-Deferoxamine Conjugates for Reducing Dietary Iron Absorption.

Hereditary hemochromatosis (HH) is a non-transfusional genetic iron overload (IO) disease wherein patients are not able to regulate dietary iron absorption, which ultimately leads to excess cellular iron accumulation. Preventative measures for HH mainly include phlebotomy and asking patients to minimize dietary iron intake. To investigate alternative iron reduction strategies, we report on prophylactic non-absorbable polymer-deferoxamine (DFO) conjugates capable of chelating and reducing excessive gut uptake of dietary iron. Three different sizes of the conjugates (56 nm, 256 nm, and 7.4 µm) were prepared, and their physicochemical properties, transit times in the gut under fed/fasted conditions, acute safety, and efficacy at reducing iron absorption in a dietary iron-overload mouse model were investigated. The conjugates were synthesized through reverse phase water-in-oil (w/o) emulsions, followed by conjugation of DFO to the resulting polymer scaffolds. In vitro studies using Caco-2 transwell assays showed that the conjugates could not permeate across the monolayer, were poorly endocytosed, and did not induce cellular toxicity. In vivo mouse studies via oral gavage demonstrated that polymer-DFO conjugates remained in the gastrointestinal (GI) tract for up to 12 h and significantly prevented escalation of serum ferritin levels and excess liver iron accumulation. Ex vivo images of the duodenum suggest that nanometer-sized conjugates (56 and 246 nm) perform better at chelating dietary iron based on longer retention times (i.e., entrapment in the villi of the duodenum) and an overall slower transit from the GI tract compared to larger micron-sized (7.4 µm) conjugates. Overall, nanometer-sized polymer-DFO conjugates were orally non-absorbable, appeared safe, and were more efficacious at reducing dietary iron absorption when taken with non-heme containing food.

Design, Synthesis, and Evaluation of DFO-Em: A Modular Chelator with Octadentate Chelation for Optimal Zirconium-89 Radiochemistry.

Zirconium-89 has quickly become a favorite radionuclide among academics and clinicians for nuclear imaging. This radiometal has a relatively long half-life, which matches the biological half-life of most antibodies, suitable decay properties for positron emission tomography (PET), and efficient and affordable cyclotron production and purification. The "gold standard" chelator for [89Zr]Zr4+ is desferrioxamine B (DFO), and although it has been used both preclinically and clinically for immunoPET with great success, it has revealed its suboptimal stability in vivo. DFO can only bind to [89Zr]Zr4+ through its six available coordination sites made up by three hydroxamic acid (HA) moieties, which is not sufficient to saturate the coordination sphere (CN 7-8). In this study, we have designed, synthesized, and characterized a new octadentate chelator we have called DFO-Em, which is an improved derivative of our previously published dodecadentate chelator DFO2. This octadentate DFO-Em chelator is smaller than DFO2 but still satisfies the coordination sphere of zirconium-89 and forms a highly stable radiometal-chelator complex. DFO-Em was synthesized by tethering a hydroxamic acid monomer to commercially available DFO using glutamic acid as a linker, providing an octadentate chelator built on a modular amino acid-based synthesis platform. Radiolabeling performance and radiochemical stability of DFO-Em were assessed in vitro by serum stability, ethylenediamine tetraacetic acid (EDTA), and hydroxyapatite challenges. Furthermore, [89Zr]Zr-(DFO-Em) and [89Zr]Zr-DFO were injected in healthy mice and measured in vivo by PET/CT imaging and ex vivo biodistribution. Additionally, the coordination of DFO-Em with Zr(IV) and its isomers was studied using density functional theory (DFT) calculations. The radiolabeling studies revealed that DFO-Em has a comparable radiolabeling profile to the gold standard chelator DFO. The in vitro stability evaluation showed that [89Zr]Zr-(DFO-Em) was significantly more stable than [89Zr]Zr-DFO, and in vivo both had similar clearance in healthy mice with a small decrease in tissue retention for [89Zr]Zr-(DFO-Em) at 24 h post injection. The DFT calculations also confirmed that Zr-(DFO-Em) can adopt highly stable 8-coordinate geometries, which along with NMR characterization suggest no fluxional behavior and the presence of a single isomer. The modular design of DFO-Em means that any natural or unnatural amino acid can be utilized as a linker to gain access to different chemistries (e.g., thiol, amine, carboxylic acid, azide) while retaining an identical coordination sphere to DFO-Em.

In Vitro and In Vivo Characterization of 89Zirconium-Labeled Lintuzumab Molecule.

Objective: Positron emission tomography (PET) imaging is a powerful non-invasive method to determine the in vivo behavior of biomolecules. Determining biodistribution and pharmacokinetic (PK) properties of targeted therapeutics can enable a better understanding of in vivo drug mechanisms such as tumor uptake, off target accumulation and clearance. Zirconium-89 (89Zr) is a readily available tetravalent PET-enabling radiometal that has been used to evaluate the biodistribution and PK of monoclonal antibodies. In the current study, we performed in vitro and in vivo characterization of 89Zr-lintuzumab, a radiolabeled anti-CD33 antibody, as a model to evaluate the in vivo binding properties in preclinical models of AML. Methods: Lintuzumab was conjugated to p-SCN-Bn-deferoxamine (DFO) and labeled with 89Zr using a 5:1 µCi:µg specific activity at 37 °C for 1h. The biological activity of 89Zr-lintuzumab was evaluated in a panel of CD33 positive cells using flow cytometry. Fox Chase SCID mice were injected with 2 × 106 OCI-AML3 cells into the right flank. After 12 days, a cohort of mice (n = 4) were injected with 89Zr-lintuzumab via tail vein. PET/CT scans of mice were acquired on days 1, 2, 3 and 7 post 89Zr-lintuzumab injection. To demonstrate 89Zr-lintuzumab specific binding to CD33 expressing tumors in vivo, a blocking study was performed. This cohort of mice (n = 4) was injected with native lintuzumab and 24 h later 89Zr-lintuzumab was administered. This group was imaged 3 and 7 days after injection of 89Zr-lintuzumab. A full ex vivo biodistribution study on both cohorts was performed on day 7. The results from the PET image and ex vivo biodistribution studies were compared. Results: Lintuzumab was successfully radiolabeled with 89Zr resulting in a 99% radiochemical yield. The 89Zr-lintuzumab radioconjugate specifically binds CD33 positive cells in a similar manner to native lintuzumab as observed by flow cytometry. PET imaging revealed high accumulation of 89Zr-lintuzumab in OCI-AML3 tumors within 24h post-injection of the radioconjugate. The 89Zr-lintuzumab high tumor uptake remains for up to 7 days. Tumor analysis of the PET data using volume of interest (VOI) showed significant blocking of 89Zr-lintuzumab in the group pre-treated with native lintuzumab (pre-blocked group), thus indicating specific targeting of CD33 on OCI-AML3 cells in vivo. The tumor uptake findings from the PET imaging study are in agreement with those from the ex vivo biodistribution results. Conclusions: PET imaging of 89Zr-lintuzumab shows high specific uptake in CD33 positive human OCI-AML3 tumors. The results from the image study agree with the observations from the ex vivo biodistribution study. Our findings collectively suggest that PET imaging using 89Zr-lintuzumab could be a powerful drug development tool to evaluate binding properties of anti-CD33 monoclonal antibodies in preclinical cancer models.

Lysine-Directed Site-Selective Bioconjugation for the Creation of Radioimmunoconjugates.

The synthesis of radioimmunoconjugates via the stochastic attachment of bifunctional chelators to lysines can yield heterogeneous products with suboptimal in vitro and in vivo behavior. In response to this, several site-selective approaches to bioconjugation have been developed, yet each has intrinsic drawbacks, such as the need for expensive reagents or the complexity of incorporating unnatural amino acids into IgGs. Herein, we describe the use of a simple and facile approach to lysine-directed site-selective bioconjugation for the generation of radioimmunoconjugates. This strategy relies upon on the selective modification of single lysine residues within each light chain of the monoclonal antibody (mAb) with a branched azide-bearing perfluorophenyl ester (PFP-bisN3) followed by the ligation of dibenzocyclooctyne (DBCO)-bearing payloads to these bioorthogonal handles via the strain-promoted azide-alkyne cycloaddition. This methodology was used to create [89Zr]Zr-SSKDFO-pertuzumab, a radioimmunoconjugate of the HER2-targeting mAb pertuzumab labeled with desferrioxamine (DFO) and the positron-emitting radiometal zirconium-89 (89Zr). [89Zr]Zr-SSKDFO-pertuzumab was compared to a pair of analogous probes: one synthesized via random lysine modification ([89Zr]Zr-DFO-pertuzumab) and another via thiol-maleimide chemistry ([89Zr]Zr-malDFO-pertuzumab). The bioconjugation strategy was assessed using ESI mass spectrometry, SDS-PAGE, and autoradiography. All three immunoconjugates demonstrated comparable binding to HER2 via flow cytometry and surface plasmon resonance (SPR), and 89Zr-labeled variants of each were synthesized in >99% radiochemical yield and molar activities of up to ∼55.5 GBq/µmol (10 mCi/mg). Subsequently, the in vivo behavior of this trio of 89Zr-immunoPET probes was interrogated in athymic nude mice bearing subcutaneous HER2-expressing BT-474 human breast cancer xenografts. [89Zr]Zr-SSKDFO-pertuzumab, [89Zr]Zr-malDFO-pertuzumab, and [89Zr]Zr-DFO-pertuzumab produced positron emission tomography (PET) images with high tumoral uptake and high tumor-to-healthy organ activity concentration ratios. A terminal biodistribution study complemented the PET results, revealing tumoral activity concentrations of 126.9 ± 50.3%ID/g, 86.9 ± 53.2%ID/g, and 92.5 ± 27.2%ID/g at 144 h post-injection for [89Zr]Zr-SSKDFO-pertuzumab, [89Zr]Zr-malDFO-pertuzumab, and [89Zr]Zr-DFO-pertuzumab, respectively. Taken together, the data clearly illustrate that this highly modular and facile approach to site-selective bioconjugation produces radioimmunoconjugates that are better-defined and more homogeneous than stochastically modified constructs and also exhibit excellent in vitro and in vivo performance. Furthermore, we contend that this lysine-directed strategy holds several key advantages over extant approaches to site-selective bioconjugation, especially in the context of production for the clinic.

FoxR is an AraC-like transcriptional regulator of ferrioxamine uptake in Salmonella enterica.

Salmonella enterica spp. produce siderophores to bind iron with high affinity and can also use three xenosiderophores secreted by other microorganisms, ferrichrome, coprogen, and ferrioxamine. Here we focused on FoxA, a TonB-dependent transporter of ferrioxamines. Adjacent to foxA is a gene annotated as a helix-turn-helix (HTH) domain-containing protein, SL0358 (foxR), in the Salmonella enterica serovar Typhimurium SL1344 genome. FoxR shares homology with transcriptional regulators belonging to the AraC/XylS family. foxR is syntenic with foxA in the Enterobacteriaceae family, suggesting their functional relatedness. Both foxA and foxR are repressed by the ferric uptake regulator (Fur) under iron-rich growth conditions. When iron is scarce, FoxR acts as a transcriptional activator of foxA by directly binding to its upstream regulatory region. A point mutation in the HTH domain of FoxR abolished this binding, as did mutation of a direct repeat motif in the foxA upstream regulatory region. Desferrioxamine (DFOE) enhanced FoxR protein stability and foxA transcription but did not affect the affinity of FoxR binding to the foxA regulatory region. In summary, we have identified FoxR as a new member of the AraC/XylS family that regulates xenosiderophore-mediated iron uptake by S. Typhimurium and likely other Enterobacteriaceae members.

In vivo tracking of [89Zr]Zr-labeled engineered extracellular vesicles by PET reveals organ-specific biodistribution based upon the route of administration.

Extracellular vesicles (EVs) have garnered increasing interest as delivery vehicles for multiple classes of therapeutics based on their role as mediators in an important, natural intercellular communication system. We recently described a platform to allow the design, production and in vivo study of human EVs with specific properties (drug or tropism modifiers). This article seeks to compare and expand upon historical biodistribution and kinetic data by comparing systemically and compartmentally administered labeled engineered EVs using in vivo and ex vivo techniques. METHODS: EVs were surface-labeled to high radiochemical purity and specific activity with 89Zirconium deferoxamine ([89Zr]Zr-DFO) and/or cy7-scrambled antisense oligonucleotide (Cy7-ExoASOscr), or luminally loaded with GFP for in vivo tracking in rodents and non-human primates (NHPs). Positron Emission Tomography (PET) and subsequent immunohistochemistry (IHC) and autoradiography (ARG) cross-validation enabled assessment of the anatomical and cellular distribution of labeled EVs both spatially and temporally. RESULTS: Over time, systemic administration of engineered EVs distributed preferentially to the liver and spleen (Intravenous, IV), gastrointestinal tract and lymph nodes (Intraperitoneal, IP) and local/regional lymph nodes (Subcutaneous, SC). Immunostaining of dissected organs displaying PET signal revealed co-localization of an EV marker (PTGFRN) with a subset of macrophage markers (CD206, F4/80, IBA1). Compartmental dosing into NHP cerebrospinal fluid (CSF) resulted in a heterogenous distribution of labeled EVs depending upon whether the route was intrathecal (ITH), intracisterna magna (ICM) or intracerebroventricular (ICV), compared to the homogeneous distribution observed in rodents. Thus anatomically, ITH administration in NHP revealed meningeal distribution along the neuraxis to the base of the skull. In contrast ICM and ICV dosing resulted in meningeal distribution around the skull and to the cervical and thoracic spinal column. Further characterization using IHC shows uptake in a subset of meningeal macrophages. CONCLUSIONS: The present studies provide a comprehensive assessment of the fate of robustly and reproducibly labeled engineered EVs across several mammalian species. The in vivo distribution was observed to be both spatially and temporally dependent upon the route of administration providing insight into potential targeting opportunities for engineered EVs carrying a therapeutic payload.

Evaluation and selection of a lead diabody for interferon-γ PET imaging.

INTRODUCTION: Interferon-γ (IFN-γ) is an appealing target to evaluate immune response in cancer immunotherapy as it is a hallmark of an active immune system. Imaging and detection via immunopositron emission tomography (immunoPET) of this soluble cytokine has been made feasible using a 89Zr-labeled (t 1/2 ~ 3.27 d) monoclonal antibody (mAb). Because of its size, using a full-length mAb as an imaging vector is not ideal for repeat serial imaging because of its prolonged blood pool residency and tumor accumulation resulting in lengthier wait times between administration and imaging. This consequently impacts the potential to image a dynamic immune response in real time. This work compares 89Zr-labeled diabodies (Db) designed with variable linker lengths between the VH and VL regions with the goal of selecting a lead Db for future studies. METHODS AND RESULTS: Four Db fragments with various linker lengths (HL-n, n = 7-13 amino acids) were each conjugated to desferrioxamine (DFO). The number of attached chelates was analyzed via mass spectrometry with all immunoconjugates exhibiting one unit of DFO attached. Db-DFO conjugates were subsequently radiolabeled with zirconium-89. All constructs radiolabeled with high yields. Each radioimmunoconjugate was tested for reactivity to IFN-γ. All tracers except for [89Zr]Zr-DFO-NCS-anti-IFN-γ HL-9 exhibited comparable immunoreactivities (>90 %) to the radiolabeled parent mAb (95.8 %). At 24 h post-labeling, the IRF values were retained except for the HL-13 construct. Imaging scans and tissue distribution studies acquired in mice bearing CT26 syngeneic colorectal tumors between 1 and 24 h post-tracer administration demonstrated variable clearance kinetics and tumor localization of each radiotracer. HL-7 had higher binding in non-tumor tissues compared to HL-11 and HL-13 at 3 h p.i. Competitive binding studies versus unmodified parent mAb (AN-18) demonstrated blocking of radiolabeled HL-11 and HL-13. [89Zr]Zr-DFO-NCS-anti-IFN-γ HL-7 was inadequately blocked. CONCLUSION: Despite nuanced differences in linker lengths, our data demonstrates that [89Zr]Zr-DFO-NCS-anti-IFN-γ HL-11 exhibited the best radiotracer properties for the assessment of IFN-γ production in vivo. Work is currently underway to test the potential of using shorter-lived isotopes, like copper-64 (t1/2 ~ 12.7 h) to match pharmacokinetics and half-lives.